Composition and methods of screening

ABSTRACT

The present invention relates to a synbiotic composition comprising a probiotic bacterial strain and a prebiotic growth medium which is specific to the growth of the probiotic bacterial strain, wherein the bacterial strain is capable of producing the same growth medium by reverse enzyme reaction. The present invention also relates to methods of producing and screening for such compositions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase of PCT/GB2014/053290, filedNov. 5, 2014, which claims priority to Great Britain Application No.1319531.8, filed Nov. 5, 2013, each of which is incorporated herein byreference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention relates to a synbiotic composition comprising a probioticbacterial strain and a prebiotic growth medium which is specific to thegrowth of the probiotic bacterial species or strain, wherein thebacterial species or strain is capable of producing the same growthmedium by reverse enzyme reaction and methods of identifying andproducing such compositions.

BACKGROUND TO THE INVENTION

Probiotics are bacteria which confer health benefits to a host.Typically, cultures of probiotic bacterial strains are consumed oradministered to individuals in order to add to and augment the naturallyoccurring bacteria population of the gut. A number of health benefitshave been associated with probiotics, including reducing the incidenceof cancer, diarrhoea and irritable bowel syndrome to name a few.Preliminary studies also indicate that probiotics can be useful inreducing serum levels of cholesterol and blood pressure and helpmodulate diabetes.

Prebiotics are dietary ingredients which can selectively enhancebeneficial indigenous gut microbiota, such as lactobacilli orbifidobacteria, and are finding much increased application into the foodsector. Prebiotics are non digestible food ingredients that areselectively metabolised by colonic bacteria which contribute to improvedhealth. As such, their use can promote beneficial changes within theindigenous gut microbial milieu and they can therefore helpsurvivability of probiotics. They are distinct from most dietary fibreslike pectin, celluloses, xylan, which are not selectively metabolised inthe gut. Criteria for classification as a prebiotic is that it mustresist gastric acidity, hydrolysis by mammalian enzymes andgastrointestinal absorption, it is fermented by intestinal microfloraand selectively stimulates the growth and/or activity of intestinalbacteria associated with health and well-being. There is no knownselective prebiotic for Lactobacilli

Fructo-oligosaccharides (FOS, inulin and oligofructose) andgalactooligosaccharides (GOS) have been demonstrated to fulfil thecriteria for prebiotic classification repeatedly in human interventionstudies.

Synbiotics are mixtures of probiotics and prebiotics that beneficiallyaffect the host by improving the survival and implantation of probioticsin the gastrointestinal tract, by stimulating the growth and/or byactivating the metabolism of one or a limited number of health-promotingbacteria, thus improving host welfare. A product containingoligofructose prebiotic and bifidobacteria probiotic could be consideredto be a synbiotic if the mixture benefitted the host. Only a fewsynbiotics products are currently known.

It is an object of the present invention to provide a synbioticcomposition which has a prebiotic component which allows for thespecific growth of a given probiotic bacterial species or strain. It isalso an object of the present invention to provide for a synbioticcomposition which incorporates a Lactobacilli component in which theprebiotic component benefits the growth of lactobacilli species. A yetfurther object of the present invention is to provide a screening methodfor identify and matching probiotic bacteria (and strains thereof) andselective prebiotic components so as to form synbiotic compositionswhich accentuate host benefits.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there isprovided a synbiotic composition comprising a probiotic bacterial strainand a prebiotic growth medium which is specific to the growth of theprobiotic bacterial genus, species or strain, wherein the bacterialstrain is capable of producing the same growth medium by reverse enzymereaction.

By utilising reverse enzyme reaction in the probiotic bacterial strainto produce a prebiotic which is specific to the probiotic and wouldtherefore act as a selective growth medium can be utilised in thesynbiotic composition which promotes the growth of the probiotic at theexpense of other bacterial strains.

The enzyme may comprise a saccharolytic or glycosidase enzymes. Thesesaccharolytic or glycosidase enzymes may be derived from bacteria orfungi. The prebiotic growth medium may comprise oligosaccharides whichmay be selected from β-galactosidases, α-galactosidases, α- andβ-glucosidases, α-mannosidases, or β-xylosidases.

Preferably, the concentration of the prebiotic growth medium is utilisedto determine the probiotic bacterial genus, species or strain.

The bacterial strain preferably comprises a Lactobacilli and maycomprises a strain selected from: Lactobacillus acidophilus,Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillusdelbrueckii ssp. bulgaricus, Lactobacillus casei, Lactobacillussalivarius, Lactobacillus salivarius ssp. salivarius, Lactobacillusfermentum, Lactobacillus reuteri or Lactobacillus helveticus.

The growth medium may comprise oligosaccharides such asgalacto-oligosacharides, (GOS), gluco-oligosacharides, orfructo-oligosaccharides (FOS) in varying concentrations. It has beenidentified in studies that if the growth medium is selective if itcomprises 20% or more GOS. Preferably, the composition or growth mediumcomprises 20% or more GOS. However, the composition or growth medium maycomprise a higher amount so that it is more specific for the desiredprobiotic bacterial genus, species or strain. For example, thecomposition or growth medium may comprise 25% or more GOS, 30% or moreGOS or 40% or more GOS. The composition or growth medium may compriseGOS in the range of 20% to 40%, 20% to 30% or 20% to 25%. It ispreferred that the oligosaccharide form is substantially the same as theform produced by β-galactosidases, α-galactosidases, α- andβ-glucosidases, α-mannosidases and β-xylosidases reverse reactions ofthe bacterial strain.

The probiotic bacterial strain will preferably be present in thecomposition in an effective amount so as to elicit a change in theproportions of the desirable indigenous gut microbiota and in particularthe probiotic bacterial strain. Preferably, the probiotic bacterialstrain is in an amount in the range of 10⁵ cfu/g to 10¹² cfu/g. Morepreferably, the probiotic bacterial strain is in an amount in the rangeof 10⁸ cfu/g to 10⁹ cfu/g. It will be appreciated that the “cfu” refersto colony forming units which is a standard measure of bacterial cellquantity. Furthermore, higher amounts may be utilised if change in themicrobiota is required quickly or if the composition is being used toseed the gut with a new bacterial strain not currently present in thebody of the human or animal to which the composition in beingadministered.

The growth medium may be present in an amount which provides optimalgrowth and survival of the probiotic bacterial strain within the gutwithout impacting on safety, tolerance, and shelf life.

The strain and/or the growth medium may be encapsulated. Manyencapsulation techniques will be apparent to the skilled addressee andthe one employed will be tailored to the required stability of theprebiotic growth medium and/or strain and desired digestive transittime. The growth medium may itself be used to encapsulate thestrain—whether entirely or within an encapsulation matrix formed of thegrowth medium and another material. It is preferred that the prebioticgrowth medium is utilised as an outer core containing the probioticbacterial strain which is specifically formulated to be released at thetarget site.

The composition may further comprise an excipient or carrier compound toenable the strain and/or growth medium to pass through thegastrointestinal environment of the body and be efficiently deliveredand released to the lower gut. The strain may be concentrated and/orfreeze or spray dried. The composition may be in a number of formats,such as a drinkable liquid and/or mixed with a solid or liquid foodstuff.

In accordance with a further aspect of the present invention, there isprovided a synbiotic composition for the treatment of a metabolicdisorder comprising a probiotic bacterial strain and a prebiotic growthmedium which is specific to the growth of the probiotic bacterial genus,species or strain, wherein the bacterial strain is capable of producingthe same growth medium by reverse enzyme reaction.

The enzyme may comprise a saccharolytic enzyme. The saccharolytic enzymemay be derived from bacteria or fungi. The prebiotic growth medium maycomprise oligosaccharides which may be selected from β-galactosidases,α-galactosidases, α- and β-glucosidases, α-mannosidases, orβ-xylosidases.

The bacterial strain preferably comprises a Lactobacilli and maycomprises a strain selected from: Lactobacillus acidophilus,Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillusdelbrueckii ssp. bulgaricus, Lactobacillus casei, Lactobacillussalivarius, Lactobacillus salivarius ssp. salivarius, Lactobacillusfermentum or Lactobacillus helveticus.

The growth medium may comprise oligosaccharides such asgalacto-oligosacharides, (GOS), gluco-oligosacharides, orfructo-oligosaccharides (FOS). It is preferred that the oligosaccharideform is substantially the same as the form produced by β-galactosidases,α-galactosidases, α- and β-glucosidases, α-mannosidases andβ-xylosidases reverse reactions of the bacterial strain.

The probiotic bacterial strain will preferably be present in thecomposition in an effective amount so as to elicit a change in theproportions of the desirable indigenous gut microbiota and in particularthe probiotic bacterial strain. Preferably, the probiotic bacterialstrain is in an amount in the range of 10⁵ cfu/g to 10¹² cfu/g. Morepreferably, the probiotic bacterial strain is in an amount in the rangeof 10⁸ cfu/g to 10⁹ cfu/g. Furthermore, higher amounts may be utilisedif change in the microbiota is required quickly or if the composition isbeing used to seed the gut with a new bacterial strain not currentlypresent in the body of the human or animal to which the composition inbeing administered.

The growth medium may be present in an amount which provides optimalgrowth and survival of the probiotic bacterial strain within the gutwithout impacting on safety, tolerance, and shelf life. Theconcentration of the prebiotic in the medium may be varied to optimisestrain, species, and genus specificity.

The strain and/or the growth medium may be encapsulated. Manyencapsulation techniques will be apparent to the skilled addressee andthe one employed will be tailored to the required stability of theprebiotic growth medium and/or strain and desired digestive transittime. The growth medium may itself be used to encapsulate thestrain—whether entirely or within an encapsulation matrix formed of thegrowth medium and another material. It is preferred that the prebioticgrowth medium is utilised as an outer core containing the probioticbacterial strain which is specifically formulated to be released at thetarget site.

The composition may further comprise an excipient or carrier compound toenable the strain and/or growth medium to pass through thegastrointestinal environment of the body and be efficiently deliveredand released to the lower gut. The strain may be concentrated and/orfreeze dried. The composition may be in a number of formats, such as adrinkable liquid and/or mixed with a solid or liquid food stuff.

The composition may be formed as a pharmaceutical or medicament whichcould treat heart disease, diabetes or obesity and other metabolicconditions.

The composition may be administered in up to two or three doses per day.It is preferable that the dosage regime will result in the maintenanceof the therapeutically effective amount of prebiotic growth medium andprobiotic bacterial strain. The dosage regime may be in conjunction witha foodstuff (including drinks) at pre-determined time points or at mealtimes.

In accordance with a yet further aspect of the present invention, thereis provided synbiotic composition for use as a dietary supplement,nutraceutical or functional food comprising a probiotic bacterial strainand a prebiotic growth medium which is specific to the growth of theprobiotic genus, species, or bacterial strain, wherein the bacterialstrain is capable of producing the same growth medium by reverse enzymereaction.

By utilising reverse enzyme reaction in the probiotic bacterial strainto produce a prebiotic which is specific to the probiotic and wouldtherefore act as a selective growth medium can be utilised in thesynbiotic composition which promotes the growth of the probiotic at theexpense of other bacterial strains.

The enzyme may comprise a saccharolytic enzyme. The saccharolytic enzymemay be derived from bacteria or fungi. The prebiotic growth medium maycomprise oligosaccharides which may be selected from β-galactosidases,α-galactosidases, α- and β-glucosidases, α-mannosidases, orβ-xylosidases.

The bacterial strain preferably comprises a Lactobacilli and maycomprises a strain selected from: Lactobacillus acidophilus,Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillusdelbrueckii ssp. bulgaricus, Lactobacillus casei, Lactobacillussalivarius, Lactobacillus salivarius ssp. salivarius, Lactobacillusfermentum or Lactobacillus helveticus.

The growth medium may comprise oligosaccharides such asgalacto-oligosacharides, (GOS), gluco-oligosacharides, orfructo-oligosaccharides (FOS). It is preferred that the oligosaccharideform is substantially the same as the form produced by β-galactosidases,α-galactosidases, α- and β-glucosidases, α-mannosidases andβ-xylosidases reverse reactions of the bacterial strain.

The probiotic bacterial strain will preferably be present in thecomposition in an effective amount so as to elicit a change in theproportions of the desirable indigenous gut microbiota and in particularthe probiotic bacterial strain. Preferably, the probiotic bacterialstrain is in an amount in the range of 10⁵ cfu/g to 10¹² cfu/g. Morepreferably, the probiotic bacterial strain is in an amount in the rangeof 10⁸ cfu/g to 10⁹ cfu/g. Furthermore, higher amounts may be utilisedif change in the microbiota is required quickly or if the composition isbeing used to seed the gut with a new bacterial strain not currentlypresent in the body of the human or animal to which the composition inbeing administered.

The growth medium may be present in an amount which provides optimalgrowth and survival of the probiotic bacterial strain within the gutwithout impacting on safety, tolerance, and shelf life. Theconcentration of the prebiotic in the medium may be varied to optimisestrain, species, and genus specificity

The strain and/or the growth medium may be encapsulated. Manyencapsulation techniques will be apparent to the skilled addressee andthe one employed will be tailored to the required stability of theprebiotic growth medium and/or strain and desired digestive transittime. The growth medium may itself be used to encapsulate thestrain—whether entirely or within an encapsulation matrix formed of thegrowth medium and another material. It is preferred that the prebioticgrowth medium is utilised as an outer core containing the probioticbacterial strain which is specifically formulated to be released at thetarget site.

The composition may further comprise an excipient or carrier compound toenable the strain and/or growth medium to pass through thegastrointestinal environment of the body and be efficiently deliveredand released to the lower gut. The strain may be concentrated and/orfreeze dried. The composition may be in a number of formats, such as adrinkable liquid and/or mixed with a solid or liquid food stuff.

Furthermore, the composition could be incorporated into an existingfood, such as yoghurt or as a powder which can be easily blended withfoodstuffs or made into a liquid drink. The composition may be combinedwith other active ingredients, such as minerals, vitamins andantioxidants.

In accordance with a further aspect of the present invention, there isprovided a method of producing a synbiotic composition comprising thesteps:

-   -   (a) selecting a probiotic bacterial strain capable of producing        a prebiotic growth medium by reverse enzyme reaction; and    -   (b) combining the bacterial strain with the growth medium so as        to form the composition.

It is preferred that the method is used for producing a composition asherein above described.

In accordance with a yet further aspect of the present invention, thereis provided a method for identifying and formulating a synbioticcomposition comprising:

-   -   (a) a first screening of a number of probiotic bacterial strains        for the ability to produce a prebiotic growth medium by reverse        enzyme reaction and identifying strains having such ability;    -   (b) a second screening of the prebiotic growth mediums of the        identified strains for the ability to be a selective growth        medium for an individual probiotic bacterial strain; and    -   (c) formulating a symbiotic composition comprising the        individual probiotic bacterial strain and selective growth        medium for that strain.

Preferably, the method is used to form a synbiotic composition as hereinabove described.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will now be described, by way ofexample only and with reference to the following Figures:

FIG. 1A-1C are graphs show the results of a range of lactobacillispecies which were screened for β-galactosidase activity measured atOD₄₂₀ in A MRS broth, B 1% lactose basal media and C 5% lactose basalmedia;

FIG. 2A-2C are graphs show the results of a range of lactobacillispecies which were screened for β-galactosidase activity measured at uMof o-NP in A MRS broth, B 1% lactose basal media and C 5% lactose basalmedia;

FIG. 3 is a graph showing the yield of GOS, lactose and monosaccharidesby L. fermentum ATCC 11976 over 168 hours;

FIG. 4 is a graph showing the yield of GOS, lactose and monosaccharidesby L. fermentum NCIMB 30226 over 168 hours;

FIGS. 5 & 6 shows graphs of the quantity of Sugars (GOS, Lactose andMonosaccharides) and GOS % over time for L. fermentum ATCC 11976;

FIGS. 7 & 8 shows graphs of the quantity of Sugars (GOS, Lactose andMonosaccharides) and GOS % over time for L. fermentum NCIMB 30226;

FIG. 9 shows graphs of the quantity of Sugars (GOS, Lactose andMonosaccharides) and GOS % over time for 18 U. L. fermentum ATCC 11976;

FIG. 10 shows graphs of the quantity of Sugars (GOS, Lactose andMonosaccharides) and GOS % over time for 18 U. L. fermentum NCIMB 30226;

FIG. 11 shows graphs of the quantity of Sugars (GOS, Lactose andMonosaccharides) and GOS % over time for 30 U. L. fermentum ATCC 11976;

FIG. 12 shows graphs of the quantity of Sugars (GOS, Lactose andMonosaccharides) and GOS % over time for 30 U. L. fermentum NCIMB 30226;

FIG. 13 is a graph illustrating the relative growth profiles of a rangeof bacteria grown on a GOS mixture produced from L. fermentum ATCC11976; and

FIG. 14 is a second graph illustrating the relative growth profiles of asmaller range of bacteria grown on a GOS mixture produced from L.fermentum ATCC 11976.

Mechanistically glycosidases are all transferases that use water astheir preferred acceptor molecule. Under appropriate circumstance,however, such as high concentrations of substrate carbohydrate, theseenzymes will transfer monosaccharide moieties from the substrate (actingas glycosyl donor) to other substrate or non-substrate carbohydrates(acting as glycosyl acceptor). Typically, the products of thesereactions are complex mixtures containing all possible glycosidiclinkages but in differing amounts. As the reactions are kineticallycontrolled, the linkage profile synthesised should map onto the rateconstants for hydrolysis of those linkages by the producing enzyme.Consequently the oligosaccharides may be more readily metabolised by theproducing organisms than by others in the gastrointestinal ecosystem.This approach has shown promise in laboratory testing.

It is possible, however in many enzyme synthesis reactions to includeother carbohydrates which will act as acceptors in addition to thelactose. In this way, novel mixtures containing novel structures couldbe built up.

Probiotic species such as lactobacilli and bifidobacteria are highlysaccharolytic and they frequently produce a range of glycosidaseenzymes. These enzymes may have transfer activity and be able tosynthesise oligosaccharides. This activity is widely reported forβ-galactosidases but has not been as intensively studied for otherenzymes such as α-galactosidases, α- and β-glucosidases, α-mannosidases,or β-xylosidases. It is also possible to synthesise oligosaccharidesusing sucrose dependant glycosyltransferases. These transfer either thefructose or glucose moiety from sucrose to sucrose acceptors and buildup long polysaccharide chains. In the presence of suitable acceptors,however, they frequently synthesise hetero-oligosaccharides. This hasbeen shown to occur with dextransucrase and alternansucrase and may alsooccur with laevansucrase.

The experiments sought to explore a strategy to use the products of onesynthesis reaction as acceptors in a subsequent reaction. If a probioticproduces a β-galactosidase and a laevan sucrase, for instance, an enzymeextract could be used to synthesise galactooligosaccharides. Thisproduct mixture could then be used with the same extract and sucrose asglycosyl donor to bring about the synthesis of fructans—many of whichwould be built up on the galacto-oligosaccharides which would act asacceptors. In this way novel complex mixtures could be produced thatshould have a highly tailored fermentation by the producing organism.

One particular experiment was conducted to reversibly useβ-galactosidases in microorganisms. Ordinarily, these would digestlactose. However, by changing the reaction conditions, in terms ofsubstrate and temperature, the enzyme acts reversibly and generates anoligosaccharide version of the lactose (GOS).

Lactobacilli are more frequently used as probiotics than arebifidobacteria, yet no prebiotic selective to lactobacilli exists. Asthese probiotics also harbour β-galactosidase activity, GOS which wasspecific to these probiotics was produced. The metabolism of prebioticslike GOS are species specific (as evidenced by Bi-Immuno and Bifidobacteria), so a Lactobacilli GOS has the potentially enhance the growth,survivability, and health benefits of lactobacilli. Ultimately, bycombining the prebiotic and probiotic an efficacious synbiotic wasgenerated (which would improve gut survival of the former).

The experiments undertaken were as follows:

-   -   1. Assemble and test a range of probiotic lactobacilli for their        capacity to generate GOS. This would involve measuring        β-galactosidase activities;    -   2. Generate a prebiotic GOS using the reverse enzyme procedure;    -   3. Scale up of the novel molecule to allow in vitro testing;    -   4. Compare survival and growth of lactobacilli in the absence        and presence of the prebiotic. This would involve a series of        ‘gut model’ experiments that test the probiotics and synbiotics;    -   5. Research the possibility for using GOS as encapsulation        material for the lactobacilli; and    -   6. Test delivery properties of the encapsulation material.

The GOS prebiotic generated by a specific strain has optimisedmetabolism not just to produce the GOS, but also to metabolise it (asits generated from a reverse enzyme procedure). The GOS can therefore beincorporated with the probiotic into a synbiotic that would create ahighly selective environment for the probiotic. As a probiotic can havea specific health benefits then a synbiotic formula which is tailored toa specific health benefit can be generated.

A screening method for identifying and formulating a synbioticcomposition in accordance with an aspect of the invention follows thesteps of:

-   -   (a) Identifying health need;    -   (b) Identifying key interjection points for probiotic action e.g        BSH activity, cholesterol assimilation & heart disease;    -   (c) Screening probiotic library using high throughput screening        methodology;    -   (d) Identifying strains with potential activity & health        benefits;    -   (e) Optimising expression of activity using fermentation        processes;    -   (f) Screening strains for glycosidase (e.g beta galactosidase)        activity;    -   (g) Generating a novel oligosaccharide (e.g GOS)    -   (h) Scaling up to allow in vitro testing;    -   (i) Comparing survival and growth of the probiotic in the        absence and presence of the prebiotic using in vitro plate        assays and gut model. If strain characterised then use molecular        methodologies to study population changes over time. This will        see if affect due to increasing number or increasing activity;        and    -   (j) Combining pre & probiotic to explore Optibiotic affect of        combined pre & probiotic.        Evaluation of Anaerobic Utilisation of Novel L. reuteri GOS

In these experiments, anaerobic cultures were tested to evaluate the invitro utilisation of a novel Lactobacillus reuterigalactooligosaccharide by monitoring the populations of gut bacterialgroups at 24 hours using fluorescent in situ hybridisation, andshort-chain fatty acid (SCFA). Fructooligosaccharides (FOS), melibioseand raffinose were used as reference carbohydrates. The table belowshows the results of these experiments.

GOS + GOS + Melibiose Raffinose FOS GOS L. acidophilus L. reuterri 24 hr% 24 hr % 24 hr % 24 hr % 24 hr 24 hr % Group Inoculum 24 change 24change 24 change 24 change 24 % change 24 change Total count 8.84 9.14103% 9.19 104% 9.2 104% 9.12 103% 9.55 108% 9.34 106% Bifidobacteria6.85 7.33 107% 7.69 112% 7.47 109% 7.69 112% 7.83 114% 8.19 120%Bacteroides 7.98 7.9 99% 8.08 101% 8.08 101% 7.95 100% 8.01 100% 7.8999% Lactobacilli 7.15 7.43 104% 7.45 104% 7.32 102% 7.69 108% 7.67 107%7.73 108% Clostridia 7.55 7.65 101% 7.81 103% 8 106% 7.23 96% 7.48 99%7.2 95% E. coli 8.14 7.66 94% 8.03 99% 7.85 96% 8.04 99% 8.24 101% 7.9698% Eubacteria 8.06 7.84 97% 8.69 108% 8.27 103% 7.75 96% 8.16 101% 8.28103% (Key: BOLD = Significant Increase; Italics = Significant Decrease)

The results show the Lactobacillus reuterri GOS showed a significantincrease in bifidobacteria and lactobacilli population numbersexhibiting a prebiotic affect. In addition, the GOS increased the growthrate of lactobacilli by 108%, more than any other sugar suggesting agenus specificity. Addition of a strain of Lactobacillus reuterriincreased the prebiotic affect, increasing the bifidobacteriumpopulation by 120%.

This suggests that the addition of a GOS producing organism to the GOSproduced by that organism had a greater effect on the gut microflorapopulation than the GOS alone.

Lactobacilli β-Galactosidase Screening Assay

In these experiments, 10 lactobacilli species were screened forβ-galactosidase activity in triplicate using standard enzyme assay witho-NPG as substrate. The experiments were carried out in 3 differentmedia; MRS, 1% and 5% lactose in basal media, as lactose is the primarysubstrate for β-galactosidase it was expected to exhibit highestactivity. Activity was measured at time points between time 0-24 hrs,highest activity was shown after 24 hrs. As shown in FIGS. 1-2, ingeneral, 5% lactose exhibits highest enzyme activity and tends to behigher than in MRS broth (contains only glucose as carbon source). Highenzyme activity is essential for generating GOS, the 3 organisms whichshow overall high activity include both L. fermentum strains and L.casei.

GOS Produced from L. fermentum ATCC 11976 and L. fermentum NCIMB 30226in a Long Time Period

In these experiments, L. fermentum ATCC 11976 and L. fermentum NCIMB30226 were assessed for their production (and consumption) of GOS,lactose and monosaccharides over 168 hours.

The yield of GOS, lactose and monosaccharides for L. fermentum ATCC11976 is shown in the below and in FIG. 3:

Time point GOS lactose Monosaccharides Total GOS %= 0 0.601 85 1.46487.065 0.690289 16 15.65 30.077 18.92 64.647 24.20839 22 183 130 75 38847.16495 36 14.4 25.6 11.45 51.45 27.98834 48 14 33 10 57 24.5614 16827.4 32.971 0.5 60.871 45.01322The yield of GOS, lactose and monosaccharides for L. fermentum NCIMB30226 is shown in the below and in FIG. 4:

Time point GOS lactose Monosaccharides Total GOS %= 0 2.206 53.309 2.53858.053 3.799976 16 20.789 74.275 24.481 119.545 17.3901 22 15.066 53.91815.713 84.697 17.78812 36 9.699 30.672 6.977 47.348 20.4845 48 13.97147.341 7.944 69.256 20.17298 168 9.3 28.125 0.521 37.946 24.50851GOS Produced from L. fermentum ATCC 11976 in a 20% Lactose Medium Over24 Hours

In this experiment, GOS synthesis from L. fermentum ATCC 11976β-galactosidase was investigated. After lysis, the crude extract wasincubated in 20% lactose over 24 hr and samples taken at time 0 and 24.

The table below shows the sugars present at TO:

Ret. Time Height Width Asym. Plates No. min v min Type (EP) (EP) 1 0.2260.397 n.a. BM n.a. n.a. 2 0.689 0.283 n.a. MB n.a. n.a. 3 6.912 1.743n.a. Ru n.a. n.a. 4 8.436 1.465 n.a. Ru n.a. n.a. 5 9.072 1.234 n.a. Run.a. n.a. 6 10.716 13.758  1.419 BMb 0.87  851 7 14.403 0.605 n.a. Run.a. n.a. 8 18.457 16.603 n.a. bM n.a. n.a. 9 18.694 17.001 n.a. M n.a.n.a. 10 22.318 0.373 n.a. Ru n.a. n.a. 11 24.168 29.345 29.609 M n.a.n.a. 12 28.157 150.287  1.544 MB n.a. 5436 Lactose n.a. n.a. n.a. n.a.n.a. n.a. n.a. Average: 19.424 10.857 0.87 3144The table below shows the sugars present at T24:

Ret. Time Height Width Resol. Asym. Plates min v min Type (EP) (EP) (EP)2.506 0.010 n.a. BMB n.a. 1.52 128 6.903 0.097 n.a. BM n.a. n.a. n.a.10.624 10.367 1.121 M 1.75 n.a. 1425 15.062 3.082 3.812 MB 2.17 n.a. 23220.868 1.220 1.268 BMB 2.66 0.65 3522 24.177 10.614 1.097 BMb 3.50 1.577869 GOS 28.167 73.205 1.207 bM n.a. 1.45 8860 Lactose 29.600 5.0092.231 M n.a. n.a. n.a. 32.806 10.232 1.873 M 1.05 n.a. 5038 Glucose34.822 8.609 2.038 M n.a. n.a. 4812 Galac- tose 41.161 0.867 n.a. M n.a.n.a. n.a. 43.560 0.590 n.a. M n.a. n.a. n.a. 46.616 0.386 n.a. M n.a.n.a. n.a. 49.693 0.107 n.a. MB n.a. n.a. n.a. 51.010 0.006 n.a. bMB n.a.n.a. n.a. 54.025 0.006 n.a. BMB 1.18 1.41 774387 54.751 0.008 n.a. BMBn.a. 1.27 48500 n.a. n.a. n.a. n.a. n.a. n.a. n.a. 7.319 1.831 2.05 1.3185477GOS Produced from L. fermentum ATCC 11976 and L. fermentum NCIMB 30226in a Short Time Period

In this experiment, GOS was produced from L. fermentum ATCC 11976 and L.fermentum NCIMB 30226 and the enzyme activity of the sugars vs the % GOSassessed over 50 hours as this was when most activity took place duringthe previous experiments.

Protocol

GOS was produced using the following protocol:

-   -   1. Set up 50 ml overnight cultures in modified MRS broth        supplemented with 2% lactose for L. fermentum ATCC 11976 and L.        fermentum NCIMB 30226;    -   2. Suspend 50 ml of overnight culture in 1 L of mMRS broth with        2% lactose;    -   3. Incubate in anaerobic cabinet at 37° C.;    -   4. L. fermentum ATCC 11976 for 14 hours;    -   5. L. fermentum NCIMB 30226 for 8 hours;    -   6. Measure OD₆₆₀;    -   7. Centrifuge cultures, 10 000 g×10 mins;    -   8. Make up 40% lactose in sodium phosphate buffer. 400 g/L;    -   9. Pour off supernatant;    -   10. Resuspend pellets in sodium phosphate buffer (50 mM, pH        6.8);    -   11. Pool pellets in 50 ml falcons;    -   12. Freeze thaw in Liquid Nitrogen×3;    -   13. French Press, 30,000 PSI, 1 pass, 5 drops/min;    -   14. Spin down lysate—15,000 g×45 min;    -   15. Pour supernatant into fresh falcon;    -   16. Carry out β gal activity assay to work enzyme        concentrations;    -   17. Incubate the free cell extract with 40% lactose/sodium        phosphate buffer;    -   18. Sample 200 μl every 2 hours over 50 hours;    -   19. Freeze samples;    -   20. Filter sterilise all samples through 0.2 μm filter;    -   21. Analyse on HPLC.        Results—GOS Production

As shown in FIGS. 5 to 8, there was a 30-45% lactose conversion and 10%GOS yield.

Enzyme Activity

A further experiment was conducted in order to ascertain the enzymeactivity (and therefore efficiency) of the GOS produced from L.fermentum ATCC 11976 and L. fermentum NCIMB 30226.

Cultures were grown for 8 hrs F, 14 hr for F* in 1 L and harvested at12,000 g×10 min. The cells were lysed and cell extract spun down 15,000g×45 min. This was then incubated at 40° C. in 40% lactose sodiumphosphate buffer+MgCl₂ with same U of enzyme/reaction and activityanalysed on an HPLC at 2 hour time points for 36 hours.

The enzyme unit calculations were as follows:

OD pre OD₄₂₀ (enzyme) OD₄₂₀ (enzyme) Enzyme Organism harvest afterfrench press after final spin U/15 ml F*1 0.83 2.4605 2.3315 18.23977F*2 0.86 1.83 3.1955 30.17002 F1 0.94 1.833 3.812 30.0665 F2 1.13 1.57396.0115 47.63684 Where F*1, F2 18 U/reaction, F*2, F1 30 U/reaction.Results

As shown in FIGS. 9 to 12, there was a 40-50% lactose conversion and15-20% GOS yield.

Lactobacilli Specificity with GOS Purity

In this experiment, GOS produced from L. fermentum ATCC 11976 used aspart of the growth media for a range of bacteria to see if this speciesspecific GOS provided any growth specificity.

GOS Synthesis

L. fermentum ATCC 11976 was grown in modified MRS supplemented with 2%lactose in 1 L cultures for 14 hours. The culture was spun down andresuspend in a sodium phosphate buffer. The cells were lysed usingliquid Nitrogen and a French Press and the lysate spun to obtain freecell extract. The free cell extract was incubated with 40% Lactose and asample taken every 2 hours over 50 hours. Samples were loaded on HPLCafter every time point for analysis.

Growth Curves 20% GOS Mixture

1% of the impure GOS produced earlier was added to 9 ml mMRS hungates.The growth of a range of organisms were on this mixture were analysed:Clostridium difficile, Bifidobacterium bifidum, Bifidobacterium longum,Lactobacillus fermentum ATCC 11976, Lactobacillus fermentum,Lactobacillus rhamnosus, Lactobacillus casei & Lactobacillusdelbrueccki. Experiments were conducted in 3 repeats in triplicate withenumeration at 0, 3, 6, 8, 16 and 24 hours.

Results

As shown in FIGS. 13 and 14, little growth was found in C. difficile,whereas the best growth was found in L. rhamnosus. The 20% GOS mixturewas generally more selective towards lactobacilli.

The forgoing embodiments are not intended to limit the scope of theprotection afforded by the claims, but rather to describe examples ofhow the invention may be put into practice.

The invention claimed is:
 1. A synbiotic composition comprising aLactobacilli probiotic bacterial strain and a prebiotic growth mediumproduced by the strain and which is specific to the growth of theprobiotic strain, wherein the composition or growth medium comprises 20%or more galacto-oligosaccharides (GOS) produced by reverseβ-galactosidase enzyme reaction of the strain, wherein the Lactobacillibacterial strain comprises a strain selected from: Lactobacillusfermentum ATCC 11976 or Lactobacillus fermentum NCIMB 30226, and whereinthe GOS comprises β1-4 linkages.
 2. The composition of claim 1, whereinthe concentration of the prebiotic growth medium is determined by theprobiotic strain.
 3. The composition of claim 1, wherein the strain isin an amount in the range of 10⁵ cfu/g to 10¹² cfu/g.
 4. The compositionof claim 1, wherein the strain and/or the growth medium is encapsulated.5. The composition of claim 4, wherein the growth medium is used toencapsulate the strain.
 6. The composition of claim 1, wherein thecomposition further comprises an excipient or carrier compound to enablethe strain and/or growth medium to pass through the gastrointestinalenvironment of the body.
 7. The composition of claim 1, wherein thestrain is concentrated and/or freeze dried.
 8. The composition of claim1, wherein the composition is in the form of a drinkable liquid and/orcan be mixed with a solid or liquid food stuff.
 9. A medicamentcomprising the composition of claim
 1. 10. A dietary supplementcomprising the composition of claim
 1. 11. A method of producing asynbiotic composition comprising the steps: (a) selecting a Lactobacilliprobiotic bacterial strain which produces a prebiotic growth mediumcomprising galacto-oligosaccharides (GOS) by reverse β-galactosidaseenzyme reaction; and (b) establishing the concentration of the prebioticgrowth medium to provide growth selectivity of the desired quantity ofthe probiotic bacterial strain; and (c) combining the bacterial strainwith the established concentration of growth medium so as to form thecomposition, wherein the composition or growth medium comprises 20% ormore galacto-oligosaccharides (GOS) produced by the strain, wherein theLactobacilli bacterial strain comprises a strain selected from:Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillusrhamnosus, Lactobacillus plantarum, Lactobacillus delbrueckii ssp.Bulgaricus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillussalivarius ssp. Salivarius, Lactobacillus fermentum or Lactobacillushelveticus, and wherein the GOS comprises β1-4 linkages.
 12. A methodfor identifying and formulating a synbiotic composition comprising: (a)a first screening of a number of Lactobacilli probiotic bacterialstrains for the ability to produce a prebiotic growth medium comprisinggalacto-oligosaccharides (GOS) by reverse β-galactosidase enzymereaction and identifying strains having such ability; (b) a secondscreening of the prebiotic growth mediums of the identified strains forthe ability to be a selective growth medium for an individual one of theidentified probiotic bacterial strains; (c) formulating a synbioticcomposition comprising the individual probiotic bacterial strain and anidentified selective growth medium for that strain, the selective growthmedium having been produced by the strain and wherein the growth mediumcomprises 20% or more galacto-oligosaccharides (GOS) and the GOScomprises β1-4 linkages.